Methods Ecol Evol. 2019;1–7. wileyonlinelibrary.com/journal/mee3  | 1© 2019 The Authors. Methods in Ecology and Evolution © 2019 British Ecological Society

Received:1August2018  |  Accepted:21February2019DOI: 10.1111/2041-210X.13173

P R A C T I C A L T O O L S

Empowering conservation practice with efficient and economical genotyping from poor quality samples

Meghana Natesh1,2* | Ryan W. Taylor3,4* | Nathan K. Truelove5 | Elizabeth A. Hadly3 | Stephen R. Palumbi3,5 | Dmitri A. Petrov3* | Uma Ramakrishnan1*

*Equal contribution.

1National Centre for Biological Sciences,TIFR,Bangalore,India2Sastra University, Tirumalaisamudram, Thanjavur, India3DepartmentofBiology,StanfordUniversity, Stanford, California4End2End Genomics LLC, Davis, California5HopkinsMarineStation,StanfordUniversity, Pacific Grove, California

CorrespondenceDmitri PetrovEmail:dpetrov@stanford.eduandUmaRamakrishnanEmail:uramakri@ncbs.res.in

Funding informationR43HG009482, Wildlife Conservation Trust grant(UR),FulbrightNehrugrant(2092/F-NAPE/2015, UR) and Wellcome Trust/DBT India Alliance (UR, IA/S/16/2/502714), SummitFoundationandGlobalGenomeInitiative at the Smithsonian Institution (NT, SRP,SteveBox)supportedconchwork.

Handling Editor: Douglas Yu

Abstract1. Moderate-tohigh-densitygenotyping(100+SNPs)iswidelyusedtodetermineand measure individual identity, relatedness, fitness, population structure andmigrationinwildpopulations.

2. However,theseimportanttoolsaredifficulttoapplywhenhigh-qualitygeneticmaterial isunavailable.Mostgenomictoolsaredevelopedforhigh-qualityDNAsources from laboratory or medical settings. As a result, most genetic data from marketorfieldsettingsislimitedtoeasilyamplifiedmitochondrialDNAorafewmicrosatellites.

3. To enable genotyping in conservation contexts, we used next-generation se-quencingofmultiplexPCRproductsfromverylow-qualityDNAextractedfromfaeces,hairandcookedsamples.Wedemonstratedutilityandwide-rangingpo-tential application in endangered wild tigers and tracking commercial trade inCaribbean queen conch.

4. We genotyped 100 SNPs from degraded tiger samples to identify individuals,discerncloserelativesanddetectpopulationdifferentiation.Co-occurringcarni-voresdonotamplify(e.g.Indianwilddog/dhole)oraremonomorphic(e.g.leop-ard).Sixty-twoSNPsfromconchfrittersandfield-collectedsampleswereusedtotestrelatednessanddetectpopulationstructure.

5. We provide proof of concept for a rapid, simple, cost-effective and scalablemethod(forbothsamplesandnumberofloci),aframeworkthatcanbeappliedtoother conservation scenarios previously limited by low-quality DNA samples.Theseapproachesprovideacriticaladvanceforwildlifemonitoringandforensics,open thedoor to field-ready testing, andwill strengthen theuseof science inpolicydecisionsandwildlifetrade.

K E Y W O R D S

conch,conservationgenetics,endangeredspeciesmonitoringgenotyping,multiplexPCR,noninvasivesamples,SNPs,tigers

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1  | INTRODUC TION

Stemming the tide of global species decline requires continuousmonitoring and nimble, adaptivemanagement to promote speciesrecovery.Effectivemonitoringreliesonidentifyingspeciespresenceand the ability to track specific individuals and their familial rela-tionships.Whilespeciesrecoveryiscriticallydependentontrackingindividuals and their dynamics locally, integrating data across the speciesrangeallowsmonitoringofgloballarge-scalethreatsinclud-ingpopulationrangereductionandillegalwildlifetrade.

Inprinciple, all of thesegoals canbeachievedviagenotypingamodestnumberoflocisuchasmicrosatellitesorsinglenucleotidepoly-morphisms(SNPs).Tostudyendangeredspeciesthatarerareandelu-sive,approachesmustbeabletoaccommodatenoninvasivesourcesofDNAsuchasfaeces,shell,feathers,hairandsaliva,whichyieldimpure,mixedand/orextremelysmallamountsofdegradedDNA.Moreover,marketsamplesgeneratedbywildlifetrademaybeprocessed,cooked,dried ormixedwith other species, again providing low-quality andoftenmixedDNA.Currentapproachestendtorequirerelativelylargeamounts of DNA (Carroll et al., 2018, nanograms of DNA) from the targetspecies,ordemandexpensiveandgenerallyinefficientenrich-mentstrategies(Snyder-Mackleretal.,2016;Chiou&Bergey,2018).Approaches designed for lower concentrationDNA samples (Krausetal.,2015)requireexpensiveandspecializedequipment.

HerewedemonstratethatamultiplexPCRapproachfollowedbynext- generation sequencing satisfies all the requirements necessary for inexpensive,fast,andeasygenotypingoflow-qualitysamples.Ourap-proachissimilartoGT-seq(Campbell,Harmon,&Narum,2014)butcanusepubliclyavailablesoftwarefordesigningprimersandcallingSNPsand targets only short fragments in order to succeed with degraded DNA.Weillustratethepowerofthismethodfortwoendangeredspe-ciesindivergentconservationcontextsandreal-lifesettings:genotypesfromfaeces,shedhairandsalivafoundonkilledpreyfromwildIndianti-gersandfromCITES-regulatedCaribbeanqueenconchimportedtotheUSandsoldinfriedfritters(methodschematicFigure1a,b).Methodsin-cludeDNAextraction,multiplexPCR,asecondbarcodingPCR,IlluminaMiSeqsequencingandbioinformaticsforSNPgenotyping.

The tiger Panthera tigris, a charismatic carnivore, is classified as endangered by the IUCN red list. Distribution across 14 countries (Goodrichetal.,2015)makesitcriticalthatlocallycollecteddatabecomparableacrosstigerrange.Genotypingofscatorhair,alongwithrapidforensictestingofconfiscatedskinsorothertradedpartscanverifyspecies,individualidentityandsourcepopulations.Wedevel-opedamultiplexprimersetfor192SNPlociandtestedthemonfae-cal,tissue,salivaandhairsamplesfromcaptiveandwildtigers.Wealsogenotypedtwosympatriccarnivores,thedhole(Cuon alpinus), andtheleopard(Panthera pardus), that may be confused with tigers whentargetingnoninvasivesamples.

Our second example illustrates the use of this approach evenwhen reference genomes are unavailable, and again highlights use in difficultsamples:inthiscasefriedconchfritters.Althoughformerlyabundant, the queen conch (Strombus gigas) was listed in CITES Appendix II in 1990, which allows for control of trade to reduce

over-exploitation.IdentifyinggeographicancestryofillegallytradedqueenconchproductsinFloridamarketswillaidconservationactionthat will allow recovery of this formerly lucrative fishery. Because themostdirectaccessto importedconch is fromthehundredsofrestaurants inFlorida,wesought techniques thatwouldallowge-notypingfromthemostabundantmenuitem,friedconchfritters.

2  | MATERIAL S AND METHODS

2.1 | Sample collection

Fortigers,multiplesamplesfrom13captivetigers(USAzoos)repre-sentingdifferentscenarioswereusedforstandardization(SupportingInformation,TableS1).Bloodandcorrespondingfaecalswabsfromcaptivetigers, (includingoneparent–offspringandonesiblingpair)werecollectedinIndia.WildtigersweresamplednoninvasivelyfromprotectedareasacrossIndia(SupportingInformation,TableS2).

Scatandsaliva(frompredatorbitesontheprey)weresampledby swabbing the sample surface withmoistened synthetic swabs(Ramón-Laca, Soriano,Gleeson,&Godoy, 2015); tipswere storedinlysisbufferin2mlmicrocentrifugetubes.Hairwassampledusingforcepsandstoredinziplockbags.DNAfromlegacyfaecalsamples(collected in alcohol in 2014) was also tested. DNA extraction (first stepinFigure1a)andquantificationaredescribedinSM1.Twoleop-ardsandonedholewere included.Mostsamplesweregenotypedintriplicate.

TissuesamplesfromlivecaughtQueenConchmantlewerecol-lectedusingasterilizedbiopsyforcepswerepreservedinRNALater.SamplesfromconchfritterspurchasedfromMiamirestaurantswerefrozenuntildissectedtoisolateanimaltissuefragments(SM3a).

2.2 | SNP Identification and filtering

2.2.1 | Tigers

We identified SNPs from whole genome sequencing of 75 tigers of wildandcaptiveoriginfromP. t. tigris, P. t. jacksoni, P. t. altaica and P. t. sumatrae subspecies (SM1a).Alignment, filteringandpruningwereusedtoidentifySNPs(SM1a).Wecalculatedminorallelefre-quencies(MAF)foreachSNPwithineachsubspecies,andretainedSNPswithMAF10,15,20,25and30%.WeidentifiedfixedSNPsthatdifferentiatedpopulations.WeprioritizedSNPswithincontigs>10,5and1MBrespectively.Weselected10differentiatingSNPsfromeachsubpopulation,and9992polymorphicSNPsfromeachoftheaforementionedMAFcut-offs,foratotalof50,000SNPs.

2.2.2 | Conch

We extracted RNA from 96 L. gigas individuals(SM3a,SupportingInformation,TableS3).Fourqueenconchindividuals(oneeachfromAruba,Belize,FloridaandSt.Eustatius)wereimportedintoTRINITYv2.2.0(Grabherretal.,2013)toassembleadenovotranscriptome(SM3a).

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480,962 SNPs were discovered by aligning 96 conch sequences fromsixpopulationsintheCaribbeantotheassembledtranscriptome(SFGpipeline-https://github.com/bethsheets/Population-Genomics-via-RNAseq/blob/docs/guide-to-assembly-scripts.md,SM3a).

2.3 | Primer design

Publically available Primer3 (Untergasser etal., 2012; amplicon size50–90bp,primersize17–25bpandTm of 60–61°C) was used to design primers for tigerSNPs.Conchprimersweresimilarlydesignedwiththesamecriteriaasabove.Noattemptwasmadetoidentifyincom-patibilitiesbetweenprimerpairs;192primerpairswereshortlistedforbothspecies(SM1b,3b).GT-seqindexesandadapterswereused.

2.4 | PCR amplification and sequencing

LibrarypreparationconsistedofaninitialmultiplexPCRreaction,asecondPCRreactiontoaddsequencingadaptersandindexes,andsamplepooling.Sample inputDNAvolumewasadjustedtoamaximumof1ngper reaction.ThemultiplexPCR simultane-ouslyamplifiedalltargetregionsforeachsampleseparately(ina96-wellplate).ThesecondPCRreactionaddedacombinationofforward (i5) and reverse (i7) Illumina indexes to uniquely identify eachsample.Thesequencinglibrarycontainedequalvolumesofeach sample'sbarcodedproduct andwas cleanedwithAmpurebeads. Sequencing of single 150bp reads was performed onIlluminaMiSeq(SM1f).Alignmentandgenotypecallingfollowed

F IGURE  1  (a)Aschematicforprotocolandapproximatetimetaken.Detailsinthemainandsupplementarytext.(b)Ontheleft,atigerdefecating(photo:HimanushuChhattani),ascrapemark(andassociatedhairsamples,photo:KaushalPatel)andatigerhunting(photo:ShantanuPrasad,associatedlickmarks).Ontheright,aconchemergingfromitsshell,afishermanholdinghiscatchandafritter

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GT-Seqforconch(SM3b)andusedstandardopensourcetoolsfortigers(SM1h).Figure1aillustratesthestepsdescribedabove.

For tigers, genotyping success, genotype concordance acrossreplicates, relatedness between pairs of individuals of known andunknownrelationship,probabilityofidentityoftheSNPpanelandpopulationstructurewereassessed(SM1h).Forconch,genotypingsuccessandgenotypeconcordanceweretestedbycomparingSNPsacrossreplicateconches.Geneticdistanceandabilitytoassigntheconchsamplestothecorrectpopulationwasestimatedbycompari-sonofthe96transcriptomesamples(SM4).

3  | RESULTS

3.1 | Tiger

Onehundredand twenty-six targets (66%of192attempted)pro-ducedthemostconsistentresults(SM2b,SupportingInformation,

Figure S1, Table S4), and were then tested on noninvasive sam-ples from wild tigers across India and zoo individuals (Figure1b,SupportingInformation,TableS2).

The126SNPpanelforwildtigershadahighoverallgenotypingsuccessrate(Figure2,SM2c).Anaverageof95SNPs(75%,range:4–114)weresuccessfullytypedacrossallsamples.WhentigerDNAwas >0.01 ng/μl (all sample types), an averageof 105SNPs (83%,range:48–114)weretyped.

Samplereplicateswerehighlyconcordantwithahighproportionofgenotypematches(n=28triplicates,genotypeconcordance,M: 0.957,range:0.757–1.0).Differentsampletypesfromthesamein-dividualalsohadhighlyconcordantgenotypes(n = 5, concordance, M: 0.97; range: 0.91–0.99). Our error rates were comparable tolowmicrosatellitegenotypingerrorrates insomestudies (Thaden,Cocchiararo,Jarausch,&Jüngling,2017)orlowerthanothernonin-vasivestudies(Valièreetal.,2007;Mondoletal.,2014).Ourproba-bilityofmisidentificationwasvanishinglylow(pIDsibs = 1.6E- 22).

F IGURE  1  (Continued)

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Theco-occurringcarnivores, leopardanddhole,couldbedistin-guishedfromtigergenotypes.Thedholetissuesamplehadpoorampli-ficationsuccess(meanacrossreplicates<10SNPs).Leopardshadhighamplification success (M: 110 SNPs), but SNPsweremonomorphicand nearly identical across two individuals (M:0.03%mismatches).

Theknownparent–offspringandsiblingpairs(captiveindividu-als, India)hadrelatednessvaluesclose toexpected (0.5,Figure3).Observed pairwise relatedness was higher within than betweenknowngenetic clusters (seeNateshetal., 2017).As expected, re-latednessamongindividualsfromasmall,isolatedpopulation(NW,Figure3)washigh.Wildindividualsfellintothreegeneticclustersasexpected(SupportingInformation,FigureS2).

3.2 | Queen conch

We tested a 192 primer pair panel on 279 conch samples from14populations, including 48 fried conch fritters from Miami, Floridarestaurants.Our SNP successwas lower for conch than tigers (SM4).Approximatelyhalf the192 conchprimerpairs failed toprovidedata,but62targetsreachedan86%successratesimilartothe126good tiger targets (Supporting Information, Figure S3). Replicatesamples shared99%–100%of their alleles, anddifferent processed

conch samples were genetically identical, suggesting recapture.Therewerenoobviouscloserelativesamongthesamples(SupportingInformation,FiguresS4andS5).Outlyingislands(Aruba,St.Eustatius)weregeneticallydifferentiatedfromthecentralCaribbeanandFlorida(FST=0.037, 0.048 respectively, Supporting Information, Table S5).Samples from the same island group (e.g. Florida, Bahamas) or thesamecoastwerenotdifferentiated.Thesepatternsparallelarecentsurvey of queen conch with microsatellites (Truelove et al., 2017).

Conch DNA from deep-fried fritters had lower success ratesthanfrombiopsies.However,successwashighenoughforindivid-ual identification and initial population comparison. Twenty-threefritterswereprobablynotQueenconch (fewer than18SNPsam-plified),eightfrittersrevealedpoorSNPamplification(average41%success)and17fritterswerecomparabletofreshsamples(77%vs86%success,SupportingInformation,FigureS3).Thesesamplesre-vealed lower (average) genetic identity to Florida populations (av-erage78.6–80.5allelesshared,SupportingInformation,FigureS6)comparedtoPuertoRico,orAndros Island(81.2–82.3,SupportingInformation, Figure S6). While positive population identificationmay require greater geographic sampling and more SNPs (fromseveralwholegenomes),ourpilotdatasuggestthatthefritterswegenotypedare less likely tobe fromtheFloridaKeysandNassau.

F IGURE  2 SNPtypingsuccess(intriplicate)forvarioustigersamplesandcontrols.FilledcellsindicatesuccessfullytypedSNPs;colourindicatessampletype.DNAconcentrationsareontheright

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Importantly, high-resolution nuclear data were readily obtainedfrom processed commercial samples to address key conservationchallenges.

4  | DISCUSSION

OurpilotdatasetsprovideproofofconceptformultiplexSNPgeno-typingofnoninvasiveandprocessedmarketsamplesfromtwospe-cieswithvastlydifferentphysiologies,ecologiesandconservationchallenges.Theapproachissuccessfulfordegraded,cooked,mixed,orsmallandlow-concentrationsamples(downto10−3 ng DNA/μl), makingitanidealtoolformonitoringindividualsunderfieldcondi-tionsorfromcommercialmarkets.

Conservation practitioners assume that genetics is expensive.However, our method is cheap, while providing rich informationimportantforconservation.Designingasimilarprotocolforanewspecies of interest would include costs for method developmentand implementation.Developmentcosts includepolymorphicSNPascertainment, primer design and synthesis. However, note thatSNPs have already been identified for many endangered and fisher-iesspecies(e.g.Steiner,Putnam,Hoeck,&Ryder,2013).IfnoSNPshavebeenidentified,practitionerscouldascertainSNPsusingwholegenome sequencing (e.g. genome assembly of reasonable quality ~$2,000,Armstrongetal.,2018)andpooledsequencingof10indi-vidualsatapproximately50×totalcoverage,~$1,000).Theupfrontcostofprimerdesignandsynthesisisbetween$1,000and$10,000(for100to1,000primerpairs,$10perprimerpair).Oncesynthe-sized,primerscanbeusedfor38,000reactions(~400plates).The

continual advance in sequencing and oligo synthesis will drive down these initial development costs.Most important and attractive toconservationists, we estimate implementation costs (for 1,000SNPs)canbeaslowas$5persample(whenprocessingseveralhun-dredsamples).

IncreasingthenumberofSNPsbeyondafewhundredcanpro-videadditionalinformation.Forthispilot,weconstrainedthenum-beroftargetedSNPs,butitshouldbepossibletotargetmanymore.Primers chosen toamplify clustersof closely locatedSNPs shouldallow detection of very recent inbreeding using long runs of homo-zygosity (Kirinetal.,2010).LinkedSNPscouldgeneratemicrohap-lotypes, particularly useful in pedigree reconstruction (Baetscher,Clemento,Ng, Anderson, &Garza, 2018). SNP panels could allowsimultaneous species, individual and diet identification for sets ofspecies, e.g. largecarnivoresandcommonprey species in Indiaorsub- Saharan Africa.

Effective monitoring of individuals, populations and species iscritical todesigning rapid conservationactionandmanagementofendangered species like tigers. Small, isolated populationswill re-quire inbreeding management, genetics-based population assess-ment and genetically informed introduction strategies. Likewise,identificationofcommercialproductsfromillegalfishing,bushmeathunting or highly processed market samples provides importantmanagement information.Ability toassaysuchsamplescouldpro-videapowerfulincentivetoenforcelocalconservationlaws.Rapidgeneticmonitoringofendangeredspeciesfromcommonlyoccurringnoninvasivesampleswillprovideadata-pathwaytospeciesrecovery.WebelievethatmultiplexPCRpresentsanexampleofsuchrapid,ac-cessible,cheapandefficienttechnologythatwillmakethispossible.

F IGURE  3 Pairwiserelatedness(PI_HAT):self:threereplicatesofeachsample;sameindividualbutdifferentsample,Sibs:siblings,P-O:parent–offspringpair,W-P:withinpopulation,B-P:betweenpopulations.TruerelatednessunknownforW-PandB-P.SamplinglocationsandcorrespondingcoloursrepresentedintheIndiamap

     |  7Methods in Ecology and Evolu onNATESH ET Al.

ACKNOWLEDG EMENTS

San Diego Zoo Global, CSC at Oakland Zoo, SF Zoo, Bronx Zoo,WCS Russia, El Paso Zoo, Performing Animal Welfare Society, WCS India, CWS India, WCT, Bannerghatta Zoo, Himanshu, Anubhab, Prachi,Aditya,Arun,EricaCalcagno, JackieGai for tiger samples/data, and forest departments of Rajasthan, Karnataka, MadhyaPradeshandAmericanZooAssociation,TigerSSPforpermissions,Awadhesh Pandit, NCBS- NGS facility, Steve Box, Steve Canty and NateCampbellforhelpwithconchscripts.

CONFLIC T OF INTERE S T

R.W.T. and End2End Genomics L.L.C. received NIH small business funding(R43HG009482)todeveloptoolstostudynon-modelspecies.

AUTHORS’ CONTRIBUTIONS

M.N., R.W.T., D.P., U.R., E.A.H., S.P. and N.T. designed the study.M.N., R.W.T. and N.T. conducted laboratory work and analyses.M.N.,R.W.T.,D.P.,U.R.,E.A.H.,S.P.wrotethepaper.

DATA ACCE SSIBILIT Y

Raw sequences: available from NCBI (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA516037); primer sequences: supplementaryfile;scriptsforprimerdesign,SNPcallingandfilteringavailableonDryad(tiger,https://doi.org/10.5061/dryad.q15pc00,Natesh2019)andGitHub(conch,https://doi.org/10.5281/zenodo.2580291).

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:NateshM,TaylorRW,TrueloveNK,etal.Empoweringconservationpracticewithefficientandeconomicalgenotypingfrompoorqualitysamples.Methods Ecol

Evol. 2019;00:1–7. https://doi.org/10.1111/2041-210X.13173